Abstract Background: The Gram-negative obligate intracellular bacterium Chlamydia trachomatis infects epithelia of the urogenital tract causing tissue damage, pelvic inflammatory disease and infertility. Chlamydiae utilize a type III secretion (T3S) system to deliver a network of effectors to host epithelial cells to establish an infection. How the T3S effectors subvert the host cell machinery and regulate the innate immune response is poorly understood. Broad, long-term objective: The objective of this proposal is to define at the molecular level how a network of Chlamydia effectors delivered during early infection regulate the host epithelial cell cytoskeleton, tissue architecture and the innate immune response in a novel three-dimensional endometrial organoid (EO) system. This new infection model is ideal for studying Chlamydia infection ? fully differentiated and diverse polarized epithelia positioned in a 3D environment, which mimics tissue architecture in vivo and betters recapitulates the organization of the cytoskeleton and interaction with immune cells. Specific aims: Aim 1 of this study is to functionally characterize the EO infection model, examine how the T3S effector TepP affects epithelial integrity and immune cell recruitment, and define cell-type-specific responses to infection. Aim 2 investigates how an early T3S effector network cooperatively subverts host cell signaling pathways to promote inclusion biogenesis, maturation and bacterial replication. Method: I will combine a targeted gene disruption technique using group II intronic insertion to generate Chlamydia T3S effector deficient strains, a three-dimensional organoid infection model, immune cell recruitment assays, and high-resolution light microscopy. Biochemical techniques including affinity purification coupled to mass-spectrometry will identify host targets of T3S effectors. Health-relatedness: Chlamydia infections are the leading cause of preventable blindness in the developing world and the most common sexually transmitted bacterial pathogen. Repeated infections lead to chronic immune cell infiltration that can result in tissue scarring and damage. Significantly, infections can lead to severe sequelae and are associated with cervical and ovarian cancer, yet the mechanisms by which Chlamydia disrupts epithelial tissue structure, function and growth are poorly understood. The proposed research utilizes cutting-edge infection models and new genetic techniques to understand how Chlamydia virulence factors target host cell factors to disrupt epithelial tissue functions and regulate the innate immune response.